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Abstract

Background

Although most HTLV infections in Spain have been found in native intravenous drug
users carrying HTLV-2, the large immigration flows from Latin America and Sub-Saharan
Africa in recent years may have changed the prevalence and distribution of HTLV-1
and HTLV-2 infections, and hypothetically open the opportunity for introducing HTLV-3
or HTLV-4 in Spain. To assess the current seroprevalence of HTLV infection in Spain
a national multicenter, cross-sectional, study was conducted in June 2009.

Results

A total of 6,460 consecutive outpatients attending 16 hospitals were examined. Overall,
12% were immigrants, and their main origin was Latin America (4.9%), Africa (3.6%)
and other European countries (2.8%). Nine individuals were seroreactive for HTLV antibodies
(overall prevalence, 0.14%). Evidence of HTLV-1 infection was confirmed by Western
blot in 4 subjects (prevalence 0.06%) while HTLV-2 infection was found in 5 (prevalence
0.08%). Infection with HTLV types 1, 2, 3 and 4 was discarded by Western blot and
specific PCR assays in another two specimens initially reactive in the enzyme immunoassay.
All but one HTLV-1 cases were Latin-Americans while all persons with HTLV-2 infection
were native Spaniards.

Conclusions

The overall prevalence of HTLV infections in Spain remains low, with no evidence of
HTLV-3 or HTLV-4 infections so far.

Keywords:

HTLV; Spain; Seroprevalence; Epidemiology; HTLV-3; HTLV-4

Background

Four different types of human T-lymphotropic viruses (HTLV), named 1-4, have been
described in humans. HTLV-1, the first human retrovirus was identified in 1980; it
is the etiological agent of adult T-cell leukemia/lymphoma (ATLL) [1] and tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM) [2]. These illnesses fortunately only affect to less than 10% of infected individuals
lifetime. HTLV-2 was identified in 1982; it has occasionally been associated with
subacute neurological syndromes resembling TSP/HAM [3] with no evidence of producing hematological malignancies [4]. Finally, HTLV-3 and HTLV-4 were described in 2005 in a few asymptomatic individuals
from Cameroon and to date no illnesses have been associated with these viral infections
[5-8].

The main routes of transmission of HTLV are from infected mothers to their newborns,
especially through prolonged breast-feeding, sexual intercourse, blood transfusion
and sharing of needles and syringes between intravenous drug users [9]. HTLV-1 has spread worldwide with estimates of 10-20 million infected people. It
is endemic in some parts of Japan, Central and South America and Sub-Saharan Africa
[10]. In contrast, HTLV-2 infection affects 3-5 million persons and is prevalent in some
Amerindian and African pygmy tribes and epidemic among injecting drug users in Western
Europe and North America [4]. In Spain, the majority of individuals HTLV positive are native Spaniards, most of
them with past history of intravenous drug use and infected with HTLV-2. In contrast,
most persons infected with HTLV-1, which is overall less prevalent, are immigrants
coming from endemic regions in Central and South America [11-13]. Until December 2009, a total of 144 cases of HTLV-1 infection and 717 of HTLV-2
infection had been recorded at the national Spanish HTLV registry [13].

Several serological surveys conducted over the last decade have monitored the prevalence
of HTLV infections in Spain [14]. In the last serosurvey conducted in year 2008, a total of 7 out of 5,742 consecutive
hospital outpatients were found to be HTLV-seroreactive (overall rate, 0.12%). No
single case of HTLV-1 infection was reported at that time [15].

Recent estimates have pointed out that the number of immigrants legally registered
as living in Spain has increased more than six-fold over the last 10 years [16]. Immigrants currently represent nearly 6 out of 47 million people in Spain. Many
of the foreigners come from regions where the presence of HTLV-1 is endemic as some
countries in Latin-America and Sub-Saharan Africa and where HTLV-3 and HTLV-4 have
been reported occasionally [8]. This wave of immigration could have modified the prevalence of HTLV infection in
Spain and the type distribution. In order to test this hypothesis, the Spanish HTLV
Group conducted a new prospective, multicenter, national serosurvey for studying HTLV
infection in year 2009.

Results

A total of 6,460 consecutive adult outpatients attended during June 2009 at 16 distinct
hospitals were screened for HTLV antibodies. The median age of the study population
was 38 years and 40% were male. Although native Spaniards represented 88% of the total
study population, 4.9% of subjects come from Latin America, 3.6% from Africa and 2.8%
from other European countries.

Nine specimens were repeatedly EIA reactive and were confirmed by Western blot as
HTLV-1 positive (n = 4) and HTLV-2 (n = 5). Another two samples exhibited EIA reactivity
close to the cut-off but could not be confirmed as HTLV positive by Western blot or
PCR testing. No PBMCs could be obtained from the four HTLV-1 positive cases, which
precluded further virological characterization of these samples.

The prevalence of HTLV found in this study was 0.14%, being 0.06% for HTLV-1 and 0.08%
for HTLV-2. Neither HTLV-3 nor HTLV-4 were found in this survey. Three of the HTLV-1
carriers had been born in Latin America (Peru, Ecuador and the Dominican Republic)
while the last one was a 31 years-old native Spanish woman who denied any significant
risk behavior for HTLV exposure, including intravenous drug use, sexual promiscuity,
transfusions or stages in Latin America. All HTLV-1 carriers but one were asymptomatic.
The woman from the Dominican Republic was newly diagnosed with mild TSP/HAM (Table
1).

In contrast to HTLV-1 cases, all 5 subjects with HTLV-2 infection identified in this
study were native Spaniards who admitted prior intravenous drug use. Moreover, all
were coinfected with HIV. None of them complained of neurological symptoms potentially
associated with HTLV-2 (Table 1).

The prevalence of HTLV infection in the current study seems to remain fairly stable
(Table 2). Moreover, no cases of HTLV-3 nor HTLV-4 have been identified so far in Spain.

Table 2. Main characteristics of the two last cross-sectional surveys of HTLV antibodies conducted
in Spain

Discussion

For several years, periodic surveillance studies have been conducted in Spain looking
for changes in the rate and distribution of HTLV infections. Overall the seroprevalence
of HTLV infection found in this study was low (0.14%) and similar to that found in
previous serosurveys carried out in Spain [14,15]. Only 9 out of 6,460 tested individuals were found to be positive for HTLV. While
HTLV-2 seems to have been present for decades among native Spanish intravenous drug
users [17], HTLV-1 seems to have been introduced more recently. The four cases identified in
the current study support that the recent big wave of immigration from HTLV-1 endemic
regions in Latin America and Africa, could have contributed to introduce HTLV-1 infection
in Spain. Moreover, the recognition of HTLV-1 in a native Spaniard with lack of any
evident risk for HTLV-1 exposure might further support that HTLV-1 is already spreading
within the native Spanish population. In other European countries, as the United Kingdom
and France, with larger and longer presence of immigrants from HTLV-1 endemic regions,
sexual transmission of HTLV-1 from foreigners to natives has already been well documented
[18].

The recognition of HTLV-1 infected persons in Spain has several clinical and public
health implications. HTLV testing should be considered for a broader number of persons
and conditions, including blood bank donors, organ transplantation, antenatal testing
or sexually transmitted diseases clinics. Close relatives of infected individuals
should be offered for HTLV testing. Given that most HTLV-1 carriers would remain asymptomatic
life long, unaware silent transmission of the virus is the most worrisome. Wider HTLV
testing may allow identification of carriers and help to reduce further transmission.
Pregnant women with HTLV-1 infection should be advised against breast-feeding, which
is the most effective way to prevent vertical HTLV-1 transmission. Moreover, physicians
must monitor periodically asymptomatic persons known to be infected with HTLV-1, in
order to facilitate early recognition of classical complications, such as TSP/HAM
or ATL. Attention to mild symptoms or laboratory abnormalities, as in one of the current
cases complaining minimal paraparesis, may permit early diagnosis and better treatment
options.

The rate of HTLV-2 infection seems to remain fairly stable in Spain and confined mainly
to former intravenous drug users and their sexual partners. As most HTLV-2 carriers
are intravenous drug users coinfected with HIV and intravenous drug use practices
have dramatically declined in Spain [19], we should expect a steadily decline in the prevalence of HTLV-2 infection in coming
years.

It is worth to note that this is the largest study carried out in Spain examining
the prevalence of HTLV infections. We are confident that the 16 hospitals that participated
in the study properly covered the whole country. However, testing was made on hospital
outpatients and therefore bias exists in terms of extrapolation of prevalence rates
to the entire population. Studies conducted in other populations, including pregnant
women, blood donors, immigrants, sexually transmitted disease clinics, would help
to define more accurately the extent of HTLV infections in Spain. Of note, despite
testing a relatively large immigrant population from Western Africa, where HTLV-3
and HTLV-4 were described originally [5-7] no single case of infection with these HTLV variants has been reported so far in
Spain. In this study, the two samples from individuals showing weak EIA reactivity
and indeterminate western blot results were negative for HTLV-3 and HTLV-4 testing.
Moreover, they had no epidemiological link with Africa, being these two women from
Venezuela and Spain, respectively. These results are in agreement with those from
the United States, where a recent report has failed to identify HTLV-3 and/or HTLV-4
infection in risk groups, including individuals harboring indeterminate HTLV western
blot patterns [20]. Although all individuals reported to date as infected with HTLV-3 or HTLV-4 have
displayed reactivity on EIA designed for HTLV-1 and HTLV-2 screening [5,21], we should acknowledge that the overall sensitivity of these tests to pick up all
HTLV-3 and/or HTLV-4 antibodies is currently unknown [22]. Thus, misdiagnosis of HTLV-3 and/or HTLV-4 in the present study might have occurred.

Conclusions

This study supports that the prevalence of HTLV infection in Spain remains stable
and low. However there is a slightly trend towards an increase in HTLV-1 mainly driven
by the large immigration from endemic regions in Latin America over the last decades.
The dramatic decline in intravenous drug use practices in recent years may result
in a steadily decline in HTLV-2 in the near future. Altogether, HTLV-1 must be expected
to take over HTLV-2 in coming years. Given that HTLV-1 is more pathogenic and that
transmission often occurs from people unaware of their infection, HTLV testing should
be considered for a broader number of persons.

Methods

All adult outpatients attending the hospitals belonging to the HTLV Spanish Network,
were invited consecutively during June 2009 to be tested for HTLV antibodies. In order
to make sampling representative, the hospitals selected were distributed geographically
across Spain and each of them recruited at least 350 samples. Every participating
centre obtained approval from the corresponding ethical committee and informed consent
was obtained from all recruited individuals.

Pools of five sera were screened for HTLV antibodies using a commercial enzyme immunoassay
(EIA) (Murex HTLV I + II, Abbott, Madrid, Spain). This strategy has been evaluated
previously and considered as appropriated for HTLV antibody testing [23]. Sera from reactive pools were re-tested individually by EIA and confirmed using
a commercial Western blot (Genelabs Diagnostics, Redwood City, CA). Band patterns
were interpreted following the HERN criteria [24]. Briefly, HTLV-1 or HTLV-2 positivity was considered when reactivity to at least
two recombinant envelope bands (rgp21 and rgp46-I or II, respectively) and the gag
band p24 were present. In samples yielding indeterminate Western blot pattern, when
possible a new blood sample was drawn and peripheral blood mononuclear cells were
obtained. Then, genetic confirmation discarded or confirmed HTLV infections using
specific primers for HTLV types 1-4. Briefly, DNA was extracted from PBMCs with the
midi spin columns from the QIAamp DNA blood Midi kit (Qiagen), using the procedure
recommended by the manufacturer. Afterwards, polymerase chain reactions (PCR) were
carried out on genomic LTR and/or pol regions, using primers and conditions described elsewhere [17,18,25]. The presence of amplicons was checked with electrophoresis on agarose gels. Finally,
medical records were retrospectively reviewed for all individuals found to be HTLV
positives.

Statistical analysis

Results are given as proportions and median values. Comparisons were made using the
chi square test, with Fisher correction when appropriated. Differences were considered
to be significant only when p values were lower than 0.05. All analyses were performed
using SPSS version 11.0 (SPSS Inc., Chicago, IL).